Geburtshilfe Frauenheilkd 2014; 74 - PO_Onko02_09
DOI: 10.1055/s-0034-1388354

Reversal of Forssman synthetase gene hypermethylation by 5-aza-2'-deoxycytidine in A2780 ovarian cancer cells

F Jacob 1, MP Hitchins 2, 3, A Fedier 1, S Nixdorf 2, NF Hacker 4, R Ward 2, V Heinzelmann-Schwarz 1, 2, 4
  • 1Universitätsspital Basel, Department Biomedizin, Basel, Switzerland
  • 2University of New South Wales, Lowy Cancer Centre, Sydney, Australia
  • 3Stanford University, Department of Medicine, Stanford School of Medicine, Stanford, United States
  • 4Royal Hospital for Women, Gynaecological Cancer Centre, Sydney, Australia

Introduction: Forssman antigen (Fs) is a pentaglycosyl ceramide glycolipid. It is synthesized from its precursor (globotetraose) by Forssman (Fs) synthetase encoded by GBGT1. We investigated the expression of GBGT1 and genes encoding globo series-related glycosyltransferases in ovarian cancer cells, normal ovarian surface epithelium cell cells, and selected tissue samples.

Material and methods: Pathway-specific RT-qPCR (profiling of ovarian cancer cell lines: A2780, TOV112D, TOV21G, IGROV1, OVCAR3, SKOV3; and normal surface epithelium cells: HOSE6.3, HOSE17.1). Western blotting (GBGT1 protein level determination). COBRA- and bisulfite sequencing (determine DNA methylation at the CpG island of GBGT1 promoter). 5-Aza treatment (reconstitution of GBGT1 expression). Flow cytometry (determination of Fs, the product of GBGT1; helix pomatia agglutinin (HPA)-staining). Tissue samples from gynecological and colon cancer were investigated.

Results: We identified GBGT1 as the most significantly differentially expressed gene (Linkage Ward clustering analysis). Except OVCAR3, GBGT1 gene and protein expression was lower in cancer compared to HOSE cells. We determined whether DNA methylation at the GBGT1 promoter accounts for differential expression. Bisulfite sequencing demonstrated hypermethylation in A2780 (95.8%) and hypomethylation of CpG island in OVCAR3 (9.5%) cells. The percentage of methylation negatively correlated with GBGT1 expression (r =-0.86). DNA Hypomethylation also associated with GBGT1 expression in colon cancer tissue samples. 5-Aza treatment increased GBGT1 mRNA and protein expression, which then increased HPA-staining, suggesting elevated presence of Fs on these cells.

Conclusion: GBGT1 expression is epigenetically regulated by DNA methylation in cancer tissue cells and cell lines, suggesting a role of GBGT1 in cancer.